Methods of diagnosis and treatment of osteoporosis

ABSTRACT

A method of detecting osteoporosis in a mammalian is disclosed herein which includes:
         a) obtaining a sample of a bone related tissue or cells; and   b) measuring the concentration of at least a marker which is either bacteria, bacteria produced factors, or HSPs. The method may further include comparing the concentration with concentrations from the same individual over a period of time or against a standard concentration. The marker may be a bacteria, a chaperone molecule, or a bacteria produced. Also provided herein is a method of treating or preventing osteoporosis caused by a bone disease which includes administering to a mammalian subject a therapeutically effective amount of a formulation which is either an HSP antigenic formulation or a bacterial antigenic formulation. The osteoporosis can be caused by a bone disease induced by bone infectious agents such as viruses, bacteria, fungi, protozoa and parasites.

This application claims priority to U.S. Ser. No. 60/263,109 entitled“Methods of Using Heat Shock Proteins for Diagnosis and Treatment ofBone Disease” filed Jan. 19, 2001 by Kai-Uwe Lewandrowski and U.S. Ser.No. 60/304,887 entitled “Methods of Diagnosis and Treatment ofOsteoporosis” filed Jul. 12, 2001 by Kai-Uwe Lewandrowski. The U.S.Government has rights to this application by virtue of a Grant from theNational Institutes of Health.

FIELD OF THE INVENTION

The present application generally relates to methods for diagnosing boneloss. More specifically, the present application relates to identifyinghumoral markers for bone loss on the basis of bacterial or mammalianmolecular chaperones.

BACKGROUND OF THE INVENTION

Osteoporosis is a systemic disorder characterized by decreased bone massand microarchitectural deterioration of bone tissue leading to bonefragility and increased susceptibility to fractures of hip, spine, andwrist. Osteopenia has been defined as the appearance of decreased bonemineral content on radiography, but the term more appropriately refersto a phase in the continuum from decreased bone mass to fractures andinfirmity. By the time the diagnosis of osteopenia is maderadiographically, significant and irreversible bone loss has alreadyoccurred. The most common cause of osteopenia is osteoporosis; othercauses include osteomalacia and the bone disease of hyperparathyroidism.

In the United States, roughly 1 in 4 women over the age of 50 hasosteoporosis. The overall prevalence of osteoporotic fractures risesdramatically in menopausal women. Bone loss is more abrupt for the firstdecade after the onset of menopause, followed by more gradual lossthereafter (Nordin, et al., “How can we prevent osteoporosis?” inOsteoporosis, Christiansen, et al., (eds). Copenhagen, Norhaven A/S,1204-1210 (1987)). With increasing age, fracture incidence increases.The frequency of hip fractures increases exponentially with age,particularly after age 70, and is more commonly seen in white women.About 32% of women who live to age 80 have hip fractures (Gallagher, etal., Clin. Orthop. 150:163-171 (1980); Melton, et al., Am. J. Epidemiol.129:1000-1011 (1989)). A woman's risk of a hip fracture equals thecombined risk of breast, uterine, and ovarian cancer, and the risk ofdying of hip fracture is equal to breast cancer mortality (Elffors,Aging (Milano) 10:191-204 (1998)). The prevalence of vertebral fracturesis 42% in women of advanced age and/or who have decreased bone mass(Melton et al., 1989). In women, a rapid rise of vertebral fractures,which is initially associated with the onset of menopause, is followedby an increase in the frequency of wrist and hip fractures due toage-related bone loss.

Osteoporosis develops less often in men than women because bone lossstarts later and progresses more slowly in men, and there is no periodof rapid hormonal change and accompanying rapid bone loss. Differencesin bone geometry and remodeling also contribute to the lower rate offractures in men. However, in the past few years, the problem ofosteoporosis in men has become recognized as an important public healthissue, particularly in light of estimates that the number of men olderthan 70 will double between 1993 and 2050 according to the US NationalOsteoporosis Foundation.

Roughly 1 in 8 men over the age of 50 years has osteoporosis. Presently,more than 2 million men in the United States are affected byosteoporosis, and another 3 million are at risk for this disease. Eachyear, men have one third of all hip fractures that occur, and one thirdof these men will not survive more than a year. The frequency of hipfracture increases exponentially with age, particularly after age 70,and 17% of men who live to age 80 have hip fractures (Gallagher et al.,1980; Melton et al., 1989). In addition to hip fracture, men also havepainful and debilitating fractures of the spine, wrist, and other bonesdue to osteoporosis.

While the damages caused by osteoporosis are severe and are sometimesfatal, no exact clinical chemical tests on blood or urine are 115abnormal in osteoporosis. Currently used techniques are generallybiochemical markers, radiography, and measurement of bone mineraldensity (BMD). The use of these techniques is limited either by cost orby accuracy reasons.

It is an object of the present invention to provide an effective meansfor evaluation of environmental and bone infectious stresses on theskeletal system.

It is a further object of the present invention to provide a means fortreating and/or preventing infectious disorders having a negative impacton the skeletal system.

SUMMARY OF THE INVENTION

A method of detecting osteoporosis in a mammal has been developed. Themethod includes the steps of a) obtaining a sample of a bone relatedtissue or cells; and b) measuring the concentration of at least a markerwhich is one of bacteria, bacteria produced factors, or heat shockproteins (HSPs). The method may further include comparing theconcentration of a first assay with concentrations of a second or moreassays from the same individual over a period of time or against astandard concentration. The marker can an HSP such as HSP 70, HSP 60,HSP 90, gp 96, cpn10, cpn20, ubiquitin or cpn 30. The marker can also bea bacteria such as Staphylococcus aureus, Porphyronionas gingivallis,Eikenella corrodens, Actinobacilus actinomycetemcomitans, Prevotellaintermedia, Campylobacter rectus, Staphylococcus epidermidis,Salnionella spp., Escherichia coli, Neisseria gonorrhoea, Neisseriameningitis, Mycobacterial tuberculosis, Haemophius inuflenzae,Pasteurella multocida, B. bronchiseptica, or Fusobacterium nucleatum.The marker may be a bacteria produced factor such as endotoxin-LPS,gapstatin, and dermonecrotic toxin (DNT). The time between assays mayextend over a period, for example, of at least about 12 hours. In oneembodiment, the concentration of HSP is measured using an immunoassay.In another embodiment, the concentration of HSP is measured using anassay for a nucleotide molecule encoding HSP.

A method of treating or preventing osteoporosis has been developed. Atherapeutically effective amount of a formulation which is either an HSPantigenic formulation or a bacterial antigenic formulation. In oneembodiment, the osteoporosis is caused by a bone disease induced by boneinfectious agents such as viruses, bacteria, fungi, protozoa orparasites. The HSP can be HSP 60, HSP 70, HSP 90, gp 96, cpn 10, cpn 20,ubiquitin, or cpn 30, or combinations thereof. The HSP can be furthercomplexed with an antigenic material or formulated in combination withan adjuvant. The antigenic material can be a peptide or a protein havingan antigenic determinant of a virus, bacteria, fungi, protozoa orparasite that induces a bone disease.

The methods disclosed herein can be practiced using a kit formedaccording to the methods disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term molecular chaperone describes a number of unrelated proteinsthat are involved in the assembly and reassembly of proteins and in thetransmembrane transport of proteins, for example, from the cytoplasminto the mitochondria. Some of these proteins are referred to as heatshock proteins (“HSPs”) or stress proteins. Representative HSPs includeHSP 70, HSP 70, gp 96, and HSP 100. These HSPs accomplish differentkinds of chaperonin functions. For example, HSP 70, located in the cellcytoplasm, nucleus, mitochondria, or endoplasmic reticulum, (Lindquist,S., et al., 1988, Ann. Rev. Genetics 22:631-677) is involved in thepresentation of antigens to the cells of the immune system, and is alsoinvolved in the transfer, folding and assembly of proteins in normalcells. HSP 70 located in the cytosol is involved in similar activities.GP 96 present in the endoplasmic reticulum is alao involved in antigenpresentation (Srivastava, P. K., et al., 1991, Curr. Topics inMicrobiology & Immun. 167:109-123).

HSPs are essential to both prokaryotic and eukaryotic cells forchaperone function during the intracellular (un)folding, assembly andtranslocation of proteins. By definition, HSP expression is elevated incells undergoing stress, such as those in damaged or inflamed tissue.

Conditions as diverse as a rise in temperature, hypoxia, irradiation,infection and exposure to toxic chemicals can all result in increasedHSP expression.

Upon exposure to a stressor, three distinct events result in a rapidchange in metabolic activities within the cell: (1) there is increasedtranscription of HSP mRNAs which are then preferentially translocated toa cytoplasm, (2) the transcription of most other mRNAs is suppressed;and (3) the normal translational activities of the ribosomes aredisrupted so that HSPs are preferentially translated. The overall resultof these events is that the cell rapidly begins synthesizing HSPs andrepresses the synthesis of other peptides. No new peptides or RNAsynthesis is necessary to activate the translation of the heat shockpeptide genes, indicating that preexisting factors, such as viral orbacterial agents, may be involved. Cell type, state of celldifferentiation, type of stressor, and the duration and intensity ofstress can affect the quantity and quality of a particular type of HSP.

Within the HSP families, individual members have a unique degree ofsequence homology. For HSP 60, the human protein has 50% sequenceidentity with the mycobacterial homologue, with a further 20%conservative substitutions, causing several areas in the human moleculeto be fully identical to the bacterial protein. Despite their highdegree of conservation, HSPs are very immunogenic. The potential of HSPsfor inducing autoimmunity has been broadly investigated. However,induction of autoimmunity was not reported in most experimental systems.Rather, in many models of experimental autoimmune diseases, developmentof resistance to subsequent induction of a disease is a common feature.

In recent years, it has become clear that HSPs are constitutivemolecules. A number of HSPs are well known proteins, includingubiquitin, immunosuppressant-binding proteins and P-glycoprotein. HSPshave been reported to induce cell and tissue behavior consonant withtheir involvement in inflammation. HSP 70 and E. coli groEL induce poresin membranes and thus may have similar actions to a wide variety oflysins and haemolysins produced by bacteria. There are now a number ofreports that HSPs from various bacteria can induce human or murine cellsto release a range of pro-inflammatory cytokines.

Efforts have been directed to develop ways to make use of HSPs. Forexample, methods of evaluating chronic exposure of a mammal to sublethallevels of pollutants by measuring HSP concentrations have been reported.See, for example, U.S. Pat. No. 5,780,246; U.S. Pat. No. 5,232,833, bothto Sanders, et al. Compositions and methods of vaccinating with HSPs toprevent diseases such as cancers have been developed. See, for example,U.S. Pat. Nos. 5,830,464; 5,948,646; and 6,030,618, all to Srivastava.

An “antigenic molecule” as used herein refers to peptides with which theHSPs are endogenously associated in vivo (e.g., in infected cells) aswell as exogenous antigens/immunogens (i.e., with which the HSPs are notcomplexed in vivo) or antigenic/immunogenic fragments and derivativesthereof.

“Specifically hybridize” as used herein refers to nucleotide moleculeswhich hybridize with the mRNA transcribed from the gene for the HSP at astringency condition wherein selected number of base-pair mismatchesresults in nonhybridization. One skilled in the art will recognize thestringency conditions for various hybridization assay formulas dependupon the constellation of temperature, ionic concentration and pH.Generally, for optimal RNA:RNA hybridization, the temperature isinversely related to the salt concentration; the pH should be held inthe range of from about 6.9 to about 7.4, e.g., for 15 nucleotidesequences (15-mer). For RNA:DNA hybridizations, similar assay conditionsapply but lower temperatures (accompanied by higher salt concentrations)are generally employed than for RNA:RNA hybridizations.

SDS-PAGE is a common tool for protein analysis. Sodium dodecyl sulfateis a detergent that coats proteins with negative charges at a constantcharge-to-mass ratio such that in an electric field the proteins wouldtravel at the same velocity in the absence of any separation matrix.However, when the charged proteins are loaded onto a porouspolyacrylamide gel and an electric field is applied, the smallerproteins are able to travel faster through the gel to the anode (+) thanlarger proteins, which encounter more resistance traveling through thegel. There are different types of stains that can be used to develop thegel, such as Coomassie blue and Silver stain. In many cases SDS-PAGE isa qualitative tool, in which the rough quantities and sizes of proteinsin a sample can be gauged by direct comparison of the band in questionto bands of a molecular weight ladder.

The Agilent Bioanalyzer™ is a micro-total analysis system (p-TAS) thatuses miniaturized chemical chips with microchannel networks fabricatedon glass, quartz, or plastic chips. A typical channel is roughly 50microns wide and 10 microns deep. The Agilent Bioanalyzer™ Protein 200Assay was developed to more efficiently characterize proteins by sizeand concentration in a sample. This microfluidic system functionssomewhat analogously to SDS-PAGE, but confers many benefits over themacro-scale analytical method. Some advantages of the Protein 200 Assayfor the Bioanalyzer™ include small sample volumes, higher throughputs ofdata/sample, reduced resource consumption and waste production, andautomation of multi-step processes.

In the Protein 200 Assay, proteins are denatured with B-mercaptoethanoland fluorescence labeled. In addition, as in SDS-PAGE, the proteins arecoated with SDS at a constant charge-to-mass ratio. Strategicallylocated electrodes create electrokinetic forces capable of moving fluidsand separating different sized proteins. The molecules are separated bythe amount of charge on the protein. Larger, more negatively chargedproteins travel toward the cathode. As labeled protein molecules migratepast a certain point in the channel, their fluorescence is detected by alaser beam. The sensitivity of the Protein 200 Assay is known to beaffected by salt concentrations in the sample buffer because at higherconcentrations smaller amounts of protein are injected into theseparation channel.

II. Diagnostic Techniques for Osteoporosis

No exact clinical chemical tests on blood or urine are abnormal inosteoporosis, but biochemical markers, radiography, and measurement ofbone mineral density (BMD) are helpful in diagnosing osteoporosis. Bonemass density testing is used to diagnose osteoporosis, and x-ray filmsare used to rule out other bone or arthritic conditions. Thin bones maybe detected on an x-ray film, but bone density testing is more accurate.

A. Bone Densitometry

Considerable progress in the development of methods for assessingskeletal bone mass now makes it possible to detect osteoporosisnoninvasively and early. Generally, osteoporosis may be detected afterfractures that occur with minimal trauma, as an incidental finding on anx-ray film, or by measurement of BMD by bone densitometry, which is alsoknown as bone density scans. Bone density scans are considered by manyas an instant snapshot of bone status. These scans, known collectivelyas BMD tests, are used to detect the amounts of bone mass in the spine,hip, wrist, hand, heel, or the entire body and to evaluate its density.Some studies have indicated that information regarding bone-mineralcontent at any anatomic site is equally valuable for predicting the riskof fracture in general (Black, et al., J. Bone Miner Res. 7:633-638(1992); Melton, et al., J. Bone Miner Res, 8, 1227-1233 (1993)), butother studies have suggested that measurements obtained at a particularsite of interest may provide the most important information for theprediction of fracture at that site (Cummings, et al., Lancet 341:72-75(1993)). Bone mineral density tests are the most sensitive and specifictests for osteopenia are essential in predicting the risk of fracture.

Several techniques are available to measure BMD non-invasively. All oftoday's x-ray based measurement systems are precise and deliverextremely low, effective radiation doses. The main advantages of anx-ray system over a radionuclide system are safe, shortened examinationtime, greater accuracy and precision limited to high resolution, andremoval of errors due to source decay correction. The variety of bonescan techniques that are widely used today include single x-rayabsorptiometry, dual energy x-ray absorptiometry (DEXA), quantitativecomputed tomography, peripheral quantitative computed tomography,radiographic absorptiometry, quantitative ultrasound, simple photonabsorptiometry, and dual photon absorptiometry. Their development hasbeen driven by the need to overcome the inherent shortcomings of plainradiography for this purpose. Although single and dual photonabsorptiometry are still available, these older techniques are rapidlybeing replaced by single x-ray absorptiometry and DEXA, their moderncounterparts.

Of the several techniques available, DEXA has become the most widelyused technique for measuring BMD because of its low radiation,availability, capacity to evaluate multiple sites, and ease of use(Genant, et al., Am J. Med. 91(suppl5B):49S-53S (1991)). Dual energyx-ray absorptiometry can measure soft-tissue composition (lean and fatmass) and bone mass or bone density at the lumbar spine, hip, andforearm, as well as total-body BMD, with greater precision and fasterscanning times than the dual-photon absortiometry (Consensus DevelopmentCenter: “Prophylaxis and treatment of osteoporosis” in Am J. Med.90:107-101 (1991)). As a screening procedure, DEXA is limited by itsrelatively high equipment cost. The accuracy of this technique has notbeen fully documented for measuring of all skeletal sites.

A standard bone mineral report consists of measurements expressed asbone mineral content (the amount of hydroxyapatite, in grams) andconverted to area density (grams per square centimeter) within theregion of interest. In addition, normal values are provided according tosex and race and are plotted according to age. Demographic data,including the clinical indications and the patient's age, sex, race,weight, and height, are also considered. To interpret a standard bonemineral report, a region of interest must be selected. To compareindividuals, the sites of measurement should be constant because thebone mineral content varies between different bones and betweendifferent regions of the same bone. The results are compared withnormative values, and standard curves of normative values are providedfor individuals of both sexes and several races. Comparison of measuredvalues with mean values for normal young or age-matched individualspermits an assessment of the risk of fracture.

The World Health Organization recently attempted to clarify definitionsand to assist clinicians in their interpretation of bone densitometryresults. According to that report, a normal value for bone mineralcontent is within 1 standard deviation (SD) of the mean value for youngadults of the same age and sex (that is, the t score is more than −1).Osteopenia is considered to be present when the value for bone mineralcontent is more than 1 SD but not more than 2.5 SDS below the mean foryoung adults (that is, the t score is less than −1 and more than −2.5).Osteoporosis is considered to be present when the value is more than 2.5SDS below the mean bone mineral content for young adults (that is, the tscore is less than −2.5) (Kanis et al., 1994). Severe osteoporosis isconsidered to be present when the value for bone mineral content is morethan 2.5 SDS below the mean for young adults and there is at least oneso-called fragility fracture (assumed to be associated with osteoporosisbecause it occurred as a result of slight trauma). Generally, the tscore is used for the diagnosis of low bone mass or osteoporosis.

Physicians should initiate therapy to reduce the patient's risk offracture on the basis of the presence or absence of risk factors forosteoporosis. Therapy should be initiated to reduce the risk of fracturein women who have a bone mineral density t score of less than −2 in theabsence of risk factors and in those who have at score of less than −1.5if other risk factors are present.

B. Biochemical Markers of Bone Turnover

A combination of markers of bone turnover can be used in a variety ofways in the clinical investigation of osteoporosis. Growing evidencesuggests that the rate of postmenopausal bone loss may be determined bybiochemical markers, such that a single biochemical assessment shortlyafter menopause, in conjunction with a bone mass measurement, may beused to identify women with high bone turnover and who are thereforelikely to sustain a high rate of bone loss. In osteoporotic patients,markers may be used to identify the subgroup of patients with high boneturnover who may benefit from a different therapeutic strategy from thatused in patients with low bone turnover. Finally, markers can be used inthe clinical investigation of new therapeutic agents to monitor theireffect and mechanism of action (Consensus Development Center 1991).

Osteocalcin is a bone-specific protein secreted by osteoblasts, thebone-forming cells, and its serum level is a sensitive marker of therate of bone formation. Other markers of bone formation include serumlevels of total and bone-specific alkaline phosphatase and serum type 1collagen propeptide. Pyridinoline and deoxypyridinoline are collagencross-links that are released into the blood and urine during thedegeneration of type 1 collagen in the process of osteoclastic boneresorption (Delmes “Clinical use of biochemical markers of boneremodeling in osteoporosis,” in Osteoporosis Christiansen, et al.,(eds). Copenhagen, Osteopress, pp 450-458 1990). Urinary excretion ofpyridinoline such as hydroxylysylpyridinoline and lysylpyridinoline hasbeen shown to be a more sensitive and specific marker of bone resorptionthan conventional markers such as urinary hydroxyproline (Uebelhart, etal., Bone Mineral, 8, 87-96 (1990)). Plasma tartrate-resistant acidphosphatase is another marker of bone resorption (Delmes, (1990)).

C. Radiographic Findings

A reduction in bone calcium content must exceed 30% to be observed withcertainty on conventional radiographs. Radiographically evident thinningof the cortices of long bones or vertebral bodies may be noted. Plainradiographs are generally inaccurate in the diagnosis of osteoporosis asthe demonstration of bone density is strongly dependent on radiographictechnique.

III. Identification of Bacterial or Viral Markers for Osteoporosis

A. Bacterial Markers for Osteoporosis

Bacterial Infections and Bone Pathology

There is a range of bacteria involved in bone pathology. The keyquestion to be addressed in these diseases is how the bacteria stimulatepathology and how they get into bone in the frost place. In infectionsof the appendicular and axial skeleton, the answer may lie in bacteriaexpressing receptors for bone matrix components. For example,Staphylococcus aureus contains receptors for fibronectin (Raja, et al.,Infect. Immun., 582593-2598 (1990); laminin (Mota, et al., Infect.Immun., 56, 1580-1584 (1988)), collagen (Patti, et al., Infect. Immun.62:152-161 (1994)), and bone sialoglycoprotein (Ryden, et al., Eur. J.Biochem. 184:33 1-336 (1989)) that presumably serve to trap blood-borneorganisms in bone. As bacteria do not invade the periodontal tissues,the accepted paradigm is that local pathology is due to the release ofsoluble bacterial virulence factors (Wilson, Sci. Prog. 78:19-34 (1995))and that this could be a general mechanism in all bone infections.

TABLE 1 Bacteria Involved in Pathological Bone Remodeling DiseaseOrganism Periodontitis Actinobacillus (Schluger et al., 1990)actinomycetemcomitans Porphyromonas gingivallis Eikenella corrodensFusobacterium nucleatum Prevotella inter-media Campylobacter rectus.Osteomyelitis Staphylococcus aureus (Jaffe, 1972; Staphylococcus Schmid,1993) epidermidis Salmonella spp. Escherichia coli, etc. Bacterialarthritis Staphylococcus aureus (Ho, 1993; Neisseria gonorrhoea Livnehet al., 1983) Neisseria meningitis Mycobacterial tuberculosis Haemophiusinfruenzae Pasteurella multocida, etc. Infected metal Staphylococcusaureus implants (Ross, 1991) Staphylococcus epidermidis

Three possibilities exist of how bacteria cause pathological bone loss:(a) bacteria directly destroy the noncellular components of bone byliberating acid and proteases, (b) bacteria promote cellular processesthat stimulate the degradation of bone, or (c) bacteria inhibit thesynthesis of bone matrix. Mechanisms (b) and (c) may be either a directeffect of components released by bacteria or a consequence of theinduction of host factors, for example, cytokines or prostaglandins thatthen act on bone cells. Mechanisms implicated in the pathology of dentalcaries are likely to be only a minor mechanism in skeletal bonepathology.

The Capacity of Bacteria and their Products to Inhibit Bone Formation

In addition to stimulating in vitro bone resorption, endotoxin-LPS hasalso been reported to inhibit bone collagen and noncoliagenous proteinsynthesis (Millar, et al., Infect. Immun., 57, 302-306 (1986)). A numberof reports have suggested that extracts of dental plaque or of culturedperiodontopathic bacteria can inhibit bone matrix synthesis (Hopps, etal., Periodontal disease: pathogens and host immune response. Hamada, etal., eds., (Quintessence Publishing Co., Ltd., Tokyo) pp. 307-320(1991); Multanen, et al., J. Clin. Periodontol. 1:729-739 (1985)).Surface-associated proteins from oral bacteria are also able to inhibitbone matrix synthesis (Meghji, et al., J. Periodontol. 63:736-742(1992)).

Certain periodontopathic bacteria produce factors that have generalantiproliferative activity (Helgeland and Nordby, 1993; Kamen 1981;Kataoka et al., 1993; Larjava et al., 1987; Meghji, et al., Arch. OralBiol., 37:637-644 (1992); Saito et al., 1993; Shenker et al., 1991).These could possibly play a role in inhibiting osteoblast proliferationand thus impair bone remodeling. These various biological activitieshave not been purified or characterized. An antiproliferative proteinfrom A. actinomycetemcomitans that is most active against humanosteoblast-like cell lines suggests some specificity for bone. This8-kDa protein (White et al., 1995), “termed gapstatin,” does not inhibitDNA synthesis directly but inhibits cell cycle progression by blockingcells in the G2 phase of the cell cycle. Kinetic studies of synchronizedcell populations reveal that gapstatin acts only on cells in S phase.This molecule may act by inhibiting the synthesis of cyclin BI, aprotein required to ensure that cells make the transition from G2 tomitosis. As bone remodeling and matrix synthesis require the continuedproduction of osteoblasts and osteoclasts, the action of gapstatin couldinhibit new bone matrix formation. Such an effect would be particularlydamaging if it were to occur in concert with molecules stimulating bonebreakdown, such as cpn60. It is possible that gapstatin could inhibitthe formation of osteoclasts.

Bordetella bronchiseptica produces a 145 kDa dermonecrotic toxin DNT)that is responsible for turbinate atrophy in swine atrophic rhinitis(Ackerman et al., 1991; Dunan et al., 1966). Histologically, the lesionsinduced by B. bronchiseptica suggest impaired osteoblastic function(Silveira et al., 1982). There is one report of the effect of DNT oncultured bone, and its effects were not particularly striking (Kiman etal., 1987). However, when added to the murine osteoblastic cell lineMC3T3-El, it caused changes in cellular architecture and potentlyinhibited (50% inhibitory concentration, 100 pg/ml) the osteoblasts'capacity to produce both alkaline phosphatase and collagen (Horiguchi etal., 1991), an action that could seriously affect bone remodeling ifreplicated in vivo. It has recently been reported that DNT is a potentstimulator of tritiated thymidine incorporation into MC3T3-El cells witha 50% effective dose of approximately 1 ng/ml. In spite of thisincorporation of label, the numbers of MC3T3-El cells in culture did notincrease. The major consequence of exposure to DNT was the appearance ofmultinucleated osteoblasts. Another cell cycle-inhibitory protein hasrecently been isolated from the periodontopathic bacterium Fusobacteriumnucleutum. This protein blocks human T lymphocytes in the mid-G, phaseof the cell cycle (Shenke and Datar, 1995). It is expected to stimulatebone resorption.

It is now becoming clear that bacteria produce a range of proteins thatare able to interfere with the mammalian cell cycle. One can suggestthat the activity of these proteins represents a new bacterial virulencemechanism. The importance of the proliferation and maturation of bonecell lineages in bone remodeling is presumably the reason that thebacterial cell cycle modulatory proteins discovered to date induce bonepathology or come predominantly from bacteria implicated in diseasesinvolving bone matrix loss.

B. Molecular Chaperone Markers for Osteoporosis

Molecular chaperones, also known as heat shock proteins (hsp), areessential to prokaryotic and eukaryotic cellular organisms through theirchaperone function during the intracellular (un)folding, assembly andtranslocation of proteins. Four main families of structurally relatedhsps are distinguished based on their molecular weights: Hsp90, Hsp70,Hsp60 and small Hsps. By definition, Hsp expression is elevated in cellsundergoing stress, such as those in damaged or inflamed tissue.Conditions as diverse as a rise in temperature, hypoxia, irradiation,infection and exposure to toxic chemicals can all result in increasedHsp expression. Within the Hsp families, individual members have aunique degree of sequence homology. For Hsp60, the human protein has 50%sequence identity with the mycobacterial homologue, with a further 20%conservative substitutions, causing several areas in the human moleculeto be fully identical to the bacterial protein.

The best studied molecular chaperones are the chaperonins (cpns) whichconsist of two interacting oligomeric proteins known as cpn10 and cpn60(from the molecular masses of their subunits). The cpns form heptamericstructures with protein folding occurring within the cavity of the cpn60oligomer, a process requiring heptameric cpn10. In contrast, the Hsp70family of molecular chaperones act as monomers. The Hsp90 family is oneof the most abundant proteins in unstressed eukaryotic cells and thisdimeric protein interacts with a large number of intracellular proteins,most notably the steroid receptors. There are also a number of lowermolecular mass molecular chaperones (Wilson, et al., J. Periodontal.Res. 20:484-491 (1985)). A number of other less well characterizedmolecular chaperones are known. Hsp47 is known to be a collagenmolecular chaperone (Laemmli, 1970). Certain of the molecular chaperonescpn60, Hsp70, and Hsp104, bind and hydrolyze adenosine triphosphate.

To isolate and further analyze HSP of a mammalian subject, tissues orcells are generally sampled under conditions which do not elevate HSPlevels. The method of tissue or cell sampling, HSP isolation andmeasurement, and formation of HSP complexes are described by U.S. Pat.Nos. 5,232,833 and 5,780,246 to Sanders, incorporated herein byreference.

Collection of Human Bone Samples from Representative Age Groups andCharacterization of the Sample Pool with Respect to Measuring BoneDensity

Representative bone samples can be obtained from a qualified bone bank.The sample pool is then characterized to generate a database based onmeasurements of bone density. Many bone density techniques have shownclinical utility for assessing fracture risk. Presently, there are morethan 20 different devices available for measurement of bone density.Some devices offer advantages in terms of versatility (i.e., the numberof skeletal sites that can be measured), ability to monitor response,cost, availability, and ease of use (Table 2). Currently, no singledevice exists that ideally addresses all of these clinical requirements.

TABLE 2 Comparison of Bone Densitometry Techniques Ease Clinical ofRadiation Method^(a) utility Versatility use Availability Cost doseSXA + − + + + + DXA ++ ++ + + − + pDXA + − + + + + QCT ++ − − + − −pQCT + − + − − + QUS + − + + + ++ ^(a)SXA, single X-ray absorptiometry;DXA, dual X-ray absorptiometry; pDXA, peripheral dual X-rayabsorptiometry; QCT, quantitative computed tomography; pQCT, peripheralquantitative computed tomography; QUS, quantitative ultrasonography.

C. Confirmation of Osteoporosis According to the WHO

For bone mineral density (BMD) measurements to be clinically useful,they need to be expressed in comparison to established normative data.All BMD manufacturers provide normative databases for this purpose.These databases are derived from bone density measurements of largegroups of both men and women of different ages and races. Comparisonsare expressed as percentage of or as the number of standard deviationsfrom the age-matched and young normal values for healthy individuals ofthe same age, sex, and race.

Percentage scores are determined with respect to either the age-matchednormal BMD (AMN) or the young normal BMD (YN) using the followingequations:

Percent  of  age  matched = [1 + (BMD − AMN/AMN)] × 100%  (Harris,  et  al.., Bone  Miner.  17:87-95(1992))Percent  of  young  normal = [1 + (BMD − YN/YN)] × 100%  (Faulkner,  et  al.., J.  Bone  Miner  Res.  11(Suppl  I):S 96(1996))

Typically, the densitometry analysis software can calculate thesepercentage values. The standard deviation (SD) scores are also usuallyprovided by the densitometry software. The age-matched SD score iscommonly referred to as the “Z-score,” whereas the young normal standarddeviation score has been labeled the “T-score.” However, differentdensitometry systems may have different names for these parameters.

The age-matched or Z-score is calculated as the difference between thepatient's BMD and the normal BMD for those of the same age, sex, andrace (AMN), divided by the SD of the normal population. This iscalculated by the densitometry system using the following equation:

Z=(BMD−AMN)/SD

The young normal or T-score is defined in a similar fashion, except theBMD difference is expressed in terms of the YN bone density:

T=(BMD−YN)/SD

For the diagnosis of osteoporosis, the WHO has defined the followingcriteria for the assessment of osteoporosis based on a BMD measurementat any skeletal site.

-   -   1. Normal: A BMD not more than 1 SD below YN (T-score=<−1).    -   2. Low bone mass (osteopenia): A BMD between 1 and 2.5 SD below        YN (T-score <−1 and >−2.5).    -   3. Osteoporosis: A BMD 2.5 or more SD below YN (T-score=/<−2.5).    -   4. Severe osteoporosis: A BMD 2.5 or more SD below young normal        (T=/<−2.5) and the presence of one or more fragility fractures.

Sample Analysis to Identify Presence of Various Bacterial and MammalianChaperons in Bone Samples.

Extraction of Chaperone Molecules from Bone Samples

All bone samples (BS) can be harvested in sterile saline, centrifuged,washed briefly in saline, and lyophilized. Chaperone molecules areremoved from the various bacteria by, for example, by gentle salineextraction as described by Wilson et al., (1985). Briefly, bone samplesare suspended in sterile saline and stirred gently at 4° C. for 1 h. Thedebris is then removed by a means such as centrifugation and the solublecomponents are dialyzed extensively against distilled water andlyophilized. The protein content of the BS can be determined by a methodsuch as described by Lowry, et al., J. Biol. Chem. 193:265-275 (1951),the carbohydrate content can be determined by a method such as describedby Dubois, et al., Anal. Chem. 28:350-356 (1956), and the nucleic acidcontent by absorption at 260/280 run. The LPS content can be measuredusing a commercial chromogenic Limulus amebocyte lysate assay, such asone market by Pyrogent, Byk-Mallinckrodt, London, UK, according to themanufacturer's instructions.

SDS-PAGE

The components of the BS can be analyzed by for example SDS-PAGE using12% gels according to a method such as one described by Laemmli, et al.,Nature (Lond.) 227:680-685 (1970). One of ordinary skill in the art willrecognize other suitable methods. Samples can be diluted to anappropriate extent such as 1:1 with sample buffer and boiled for aperiod such as 5 min. before loading. Gels can be run using for examplea MiniProtean II system (Bio Rad Laboratories) and stained with forexample Coomassie brilliant blue (Sigma Immunochemicals). The molecularweight markers can be based on Dalton standards (Sigma Immunochemicals)or any other standards which one of ordinary skill in the art canrecognize. Gels can also be silver stained using a commercial kit suchas one marketed by Gelcode© mark silver stain kit; Pierce, Rockford,Ill., to detect both the presence of protein and carbohydrate.

Two-Dimensional PAGE

Two-dimensional PAGE gels can be run using a method such as onedescribed by O'Farrell, et al., J. Biol. Chem. 250:4007-4021 (1975).Gels can be run using for example a MiniProtean™ II system and stainedwith, for example, Coomassie blue, with similar molecular weight markersas above. The first dimension, isoelectric focusing, can be over a pHrange of 1-14, preferably 3-10. Second dimension separation can becarried out by, for example, molecular mass separation using a 12%SDS-PAGE gel.

Immunoblotting

Samples separated on one- or two-dimensional SDS-PAGE can beelectroblotted onto membranes such as hnmobilon P polyvinyldifluoridemembranes marketed by Millipore Corp., Bedford, Mass. overnight(Laemmli, 1970). Membranes can be washed with, for example, PBScontaining 0.1% Triton X-100 (Sigma Immunochemicals)(PBS-T) and blockedwith PBS-T containing 2% FCS (blocking buffer) (SeraLab). Blockedmembranes can then be incubated with the test antibody (in blockingbuffer) for a period such as 1 h and washed with buffer, for example,PBS-T. Bound test-antibody (anti-mouse) IgG can be detected using forexample peroxidase labeled goat anti-mouse IgG (gamma-chain specific)(Sigma Immunochemicals) at 1:1,000 in PBS-T2% FCS. After a final wash,the blots can be developed with a solution such as 1 mg/ml 3,3prime-diaminobenzidine tetrahydrochloride (Sigma Immunochemicals) in 50mM Tris (Sigma Immunochemicals), pH 7.6, containing 150 mM NaCl (BDI-I)and 0.05% hydrogen peroxide (Sigma Immunochemicals). Each reaction canbe terminated by extensive rinsing with distilled water.

Protein Purification

Crude BS can be fractionated using column chromatography such asfractionation at 4° C. on a Q-Sepharose™ anion exchange column (50 cmtimes 1.6 cm). The column can be equilibrated in a solution such as 20mM Tris-HCl, pH 8.5 (buffer A). The BS (generally 100-400 mg) can beloaded on in the same buffer. The column can be washed with a solutionsuch as 500 ml of buffer A and then eluted with for example 1,000 mllinear gradient of O-2 M NaCl in buffer A. Fractions can be collected,whose absorbance can be monitored at 280 nm. The location of theosteolytic chaperone protein can be determined by a combination ofactivity assay, for example, SDS-PAGE, and the Western blot analysis.Fractions containing osteolytic activity can be dialyzed against forexample deionized water to remove salt and lyophilized. The fractionwith the highest specific activity and the least number of protein bandson SDS-PAGE can then be further fractionated at room temperature on asecond column such as an anion exchange column.

The purity of the fractions can again be assessed, visually by SDS-PAGEor using another means recognizable by one of ordinary skill in the art,and 100 pg of the cleanest fraction can be dialyzed against 50 mM ofTris but &, pH 7.6, containing 10 mM KCl and 10 mM MgC12 (buffer C).This sample can be run on for example a 5-ml ATP-Sepharose™ (SigmaImmunochemicals) column. The column can be washed with buffer C andbound protein eluted in 5 mM ATP (Sigma Immunochemicals), also in bufferC. Protein can be located by SDS-PAGE and visualized using a silverstain kit (Sigma Immunochemicals). Gel filtration can be used todetermine the molecular mass range of the osteolytic chaperone proteinisolated by a method such as ATP-affinity chromatography. This can beachieved by running the purified protein on a column such as Bio-SilTSK250 (Bio Rad Laboratories) column in a buffer such as 0.1 M sodiumphosphate buffer, pH 6.7, and measuring the absorption of the collectedfractions at a wavelength in the range between 205 and 280 nm.

Data Analysis and Correlation to Clinical Outcome Variables

The experimental findings and clinical bone densitometry and Q-CTmeasurements can be documented using, for example, a standardizedrelational computer database using a numerical code system. In oneembodiment, the computer program SPSS/PC+9.0 (SPSS Inc., 44 N. MichiganAve., Chicago, Ill. 60611) is used for statistical analysis of thecompiled data. Descriptive statistics of the raw data can be carried outusing frequency tables. Normal distribution can be determined by fittingto normality and by obtaining normal probability plots, where the rankedobserved residuals (deviation from the mean) are plotted on the x-axisagainst the standardized values of the normal distribution on they-axis. Normal distribution can then be indicated if the observedresiduals fall onto the straight line. Significant differences betweenmeans can be evaluated using a T-test for dependent and independentsamples and one-way ANOVA analysis of variance. In case of astatistically significant F-Test from an ANOVA using multivariatevariables, the contributing means can be differentiated by post-hoccomparison using a Tukey HSD test for unequal samples sizes. The lattertwo tests can determine the occurrence of the various ChaperoneMolecules and the presence of an osteopenic bone sample according to apatient's age and gender and other concomitant medical conditions. Thepredominant chaperone molecule correlating with osteopenia can thus beidentified.

The relationships between osteopenia, age, gender, other contributingfactors, and the presence of chaperone molecules can be establishedusing, for example, a cross tabulation method by generating multiple-wayfrequency tables. For each possible combination of these variables,these tables yield a cell frequency, i.e. the number of cases in thepatient population that had this particular combination. In addition,such a cross tabulation method allows to evaluate the reliability of thetest of these relationships. Hence, the presence of osteopenia, age,gender, other contributing factors such as medical conditions arecategorical variables. Multiple simultaneous relations and interactionsbetween the variables of the multipleway frequency table can be examinedon the basis of log-linear equations, which allow computation of thecell frequencies that would have been expected if the variables involvedwere unrelated. This can be performed using for example an iterativeproportional fitting procedure. Thus, significant deviations of theobserved from the expected frequencies can reflect a statisticallysignificant relationship between a specific chaperone molecule andosteopenia. Significance testing of deviations of the observed from theexpected frequencies can be performed via a Pearson Chi-square test. Theresidual frequencies can be calculated by subtracting the expectedfrequencies from the observed frequencies. If no relationship exists,all residual frequencies are expected to consist of positive andnegative values of similar magnitude and to be evenly distributed acrossthe cells of the frequency table. Plotting the residuals is thereforeused as another means of assessing correlations between the presence ofchaperone molecules with osteopenia. In all statistical tests employedin this study, a significance level such as one of p<0.05 can be chosen.One of ordinary skill in the art can choose a proper significance level.

IV. Diagnosing Osteoporosis using Bacterial and/or Molecular ChaperonMarkers

The method of diagnosing osteoporosis disclosed herein generallyincludes 1) sampling the tissue or cells of a mammalian subject, 2)measuring the level of a marker and 3) designating the mammalian subjectas having osteoporosis if the level of the marker is higher than astandard level of the marker in a member of a control group. The methodmay optionally include a step of isolating the marker. The control groupis selected according to factors such as geographical location, genderand/or age. Alternatively, two or more measurements of the marker in thesame mammalian subject are made and compared during a course rangingfrom several hours for example 6 hours, several days for example 10days, to several months or several years. If the level of the marker ofthe latter measurement or assay is higher than the level of the markerin the previous measurement or assay, the mammalian subject isdesignated as having osteoporosis. Sometimes, when desirable, the twomethod of diagnosing osteoporosis can be used in combination.

Preferably, the mammalian subject is a human being. Most preferably, themammalian subject is a postmenopausal female.

The marker can be either a bacteria, a bacteria produced factor, or achaperon molecule. In one embodiment, the marker is a bacteria.Representative bacterial markers are: Actinobacillusactinomycetemcomitans, Porphyromonas gingiuallis, Eikenella corrodens,Fusobacterium nucleatum, Prevotella inter-media, Campylobacter rectus,Staphylococcus aureus, Staphylococcus epidermidis, Salmonella spp.,Escherichia coli, Neisseria gonorrhoea, Neisseria meningitis,Mycobacterial tuberculosis, Haemophius infruenzae, Pasteurellamultocida.

In another embodiment, the marker can be a chaperon molecule.Representative chaperon molecular markers are: HSP 70, HSP 70, gp 96,cpn10, and cpn20, alone or in combination. Preferably, the marker is ahuman HSP.

In still another embodiment, the marker can be a bacteria producedfactor such as endotoxin-LPS, gapstatin, or dermonecrotic toxin (DNT).

V. Methods of Treating or Preventing Osteoporosis

A. Preparation and Purification of HSP Reagents

Preparation and purification of HSP proteins and their respectivepeptide complexes are within the knowledge in the art. The complexes canbe intracellularly produced complexes having HSPs from a selectedrecombinant host cell and antigenic peptides expressed from cDNAs of adiseased bone or tissue cell; the antigenic peptides of the complex arethus representative of antigenic peptides found in such bone cell.Generally, the methods of preparing HSP complexes include the steps ofobtaining (e.g., isolating) diseased bone or other tissue cells from oneor more individuals, preparing RNA from the cells, making cDNA from theRNA, introducing the cDNA into host cells, culturing the host cells sothat the diseased bone cell-derived cDNAs are expressed, and purifyingHSPs-peptide complexes from the host cells.

The cDNA prepared from disease bone or tissue cell RNA, herein referredto as “diseased bone cDNA”, is optionally amplified prior tointroduction into a host cell for expression. The cDNAs are optionallyinserted into a cloning vector for replication purposes prior toexpression. The cDNAs are inserted into an expression vector orintrachromosomally integrated, operatively linked to regulatoryelement(s) such as a promoter, for purposes of expressing the encodedproteins in suitable host cells in vitro. The cDNAs are introduced intohost cells where they are expressed by the host cells, thereby producingintracellularly noncovalent complexes of HSPs and peptides. Therecombinant host cells can be cultured on a large scale for productionof large amounts of the immunogenic complexes. The diseased bone cDNAlibrary can be stored for future use (e.g., by lyophilization orfreezing), or expanded by replication in a cloning vector in suitablehost cells to meet increased demand for the immunogenic complexes.

The immunogenic compositions prepared from the host cells expressing thediseased bone cDNAs comprise complexes of HSPs of the host cellnoncovalently associated with peptides, inter alia, those derived fromthe diseased bone cells from which the RNA was originally derived. Suchcomplexes can induce an immune response in a patient against thediseased bone cells that is therapeutically or prophylacticallyefficacious. Preferably, the patient is the subject from whom thediseased bone cells used to make cDNA were obtained. Alternatively, thediseased bone cells can be from one or more subjects different from thepatient but having diseased bone of the same tissue type.

Optionally, host cells for expression of the diseased bone cDNAs canalso be genetically engineered to coexpress recombinantly one or moreHSP genes so that increased amounts of complexes comprising immunogenicpeptides noncovalently associated with a HSP can be produced.

The preparation and purification of HSP proteins, their respectivepeptide complexes, and isolation of antigenic/immunogenic components aredisclosed in U.S. Pat. Nos. 5,830,464; 5,948,646; and 6,030,618 toSrivastava (“the Srivastava patents”). The Srivastava patents alsodescribed the in vitro production of HSP-antigenic molecule complexesand the proper procedure for determination of immunogenicity ofHSP-peptide complexes. The Srivastava patents, and the referencesincluded therein, are incorporated by reference herein.

In a preferred embodiment, the HSP-antigenic molecule complex isautologous to the individual; that is, the complex is isolated fromeither the infected cells of the individual himself (e.g., preferablyprepared from infected tissues of the patient). Alternatively, thecomplex is produced in vitro (e.g., wherein a complex with an exogenousantigenic molecule is desired). Alternatively, the HSP and/or theantigenic molecule can be isolated from the individual or from others ormade by recombinant production methods using a cloned HSP originallyderived from the individual or from others. Exogenous antigens andfragments and derivatives (both peptide and non-peptide) thereof for usein complexing with HSPs, can be selected from among those known in theart, as well as those readily identified by standard immunoassays knownin the art by the ability to bind antibody or major histocompatibilitymolecules (MHC molecules) (antigenicity) or generate immune response(immunogenicity). Complexes of HSPs and antigenic molecules can beisolated from infected tissue of a patient, or can be produced in vitro(as is necessary in the embodiment in which an exogenous antigen is usedas the antigenic molecule). The HSP-antigenic molecule complex that isadministered to the patient can be the same or different from theHSP-antigenic molecule complex used to sensitize the APC that areadministered to the patient. In a specific embodiment wherein the APCand complexes are administered concurrently, the APC and purifiedHSP-antigenic molecule complexes can be present in a single composition,or different compositions, for administration.

HSPs that can be used include but are not limited to, HSP 70, HSP 70, gp96, cpn10, and cpn20, alone or in combination. Preferably, the HSPs arehuman HSPs. Although the HSPs can be allogeneic to the patient, in apreferred embodiment, the HSPs are autologous to (derived from) thepatient to whom they are administered. The HSPs and/or antigenicmolecules can be purified from natural sources, chemically synthesized,or recombinantly produced.

The immunogenic HSP-peptide complexes disclosed herein may include anycomplex containing an HSP and a peptide that is capable of inducing animmune response in a mammal. The peptides are preferably noncovalentlyassociated with the HSP. Preferred complexes may include, but are notlimited to, HSP 60-peptide, HSP 70-peptide and HSP 70-peptide complexes.For example, an HSP called gp 96 which is present in the endoplasmicreticulum of eukaryotic cells and is related to the cytoplasmic HSP 70scan be used to generate an effective vaccine containing a gp 96-peptidecomplex.

The compositions comprising HSP noncovalently bound to antigenicmolecules can be administered to elicit an effective specific immuneresponse to the complexed antigenic molecules (and not to the HSP). TheHSP-antigenic molecule complexes are preferably purified to at least70%, 80% or 90% of the total mg protein. In another embodiment, theHSP-antigenic molecule complexes are purified to apparent homogeneity,as assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Immunogenic or antigenic peptides that are endogenously complexed toHSPs or MHC antigens can be used as antigenic molecules for treatingand/or preventing bone diseases. For example, such peptides may beprepared that stimulate cytotoxic T cell responses against differentviral proteins including, but not limited to, proteins ofimmunodeficiency virus type I (HIV-I), human immunodeficiency virus typeII (HIV-II), hepatitis type A, hepatitis type B, hepatitis type C,influenza, Varicella, adenovirus, herpes simplex type I (HSV-I), herpessimplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus,respiratory syncytial virus, papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus,mumps virus, measles virus, rubella virus and polio virus.

In another embodiment the HSP-antigenic molecule complex to be used is acomplex that is produced in vivo in cells. Alternatively, in anembodiment wherein one wishes to use antigenic molecules by complexingthem to HSPs in vitro, HSPs can be purified for such use from theendogenous HSP-peptide complexes in the presence of ATP or low pH (orchemically synthesized or recombinantly produced). The procedures forpurification of HSPs and their respective complexes are described in theSrivastava patents. The protocols described herein may be used toisolate HSP-peptide complexes, or the HSPs alone, from any eukaryoticcells for example, tissues, isolated cells, or immortalized eukaryotecell lines infected with a preselected intracellular pathogen.

B. Exogenous Antigenic Molecules

Exogenous antigens or antigenic portions can be selected for use asantigenic molecules, for complexing to HSPs, from among those known inthe art or determined by immunoassay to be able to bind to antibody orMHC molecules (antigenicity) or generate immune response(immunogenicity). To determine immunogenicity or antigenicity bydetecting binding to antibody, various immunoassays known in the art canbe used, including but not limited to competitive and non-competitiveassay systems using techniques such as radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays, immunoradiometricassays, gel diffusion precipitin reactions, immunodiffusion assays, invivo immunoassays (using colloidal gold, enzyme or radioisotope labels,for example), western blots, immunoprecipitation reactions,agglutination assays (e.g., gel agglutination assays, hemagglutinationassays), complement fixation assays, immunofluorescence assays, proteinA assays, and immunoelectrophoresis assays, etc. In one embodiment,antibody binding is detected by detecting a label on the primaryantibody. In another embodiment, the primary antibody is detected bydetecting binding of a secondary antibody or reagent to the primaryantibody. In a further embodiment, the secondary antibody is labelled.Many means are known in the art for detecting binding in an immunoassayand are envisioned for use. In one embodiment for detectingimmunogenicity, T cell-mediated responses can be assayed by standardmethods, e.g., in vitro cytoxicity assays or in vivo delayed-typehypersensitivity assays.

Potentially useful antigens or derivatives thereof for use as antigenicmolecules can also be identified by various criteria, such as theantigen's involvement in neutralization of a pathogen's infectivity(wherein it is desired to treat or prevent infection by such a pathogen)(Norrby, 1985, Summary, in Vaccines 85, Lerner, et al. (eds.), ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., pp. 388-389), typeor group specificity, recognition by patients' antisera or immune cells,and/or the demonstration of protective effects of antisera or immunecells specific for the antigen. In addition, where it is desired totreat or prevent a disease caused by pathogen, the antigen's encodedepitope should preferably display a small or no degree of antigenicvariation in time or amongst different isolates of the same pathogen.Preferably, where it is desired to treat or prevent osteoporosis causedby viral infections of bone, molecules comprising epitopes of knownviruses are used, as discussed above. Preferably, where it is desired totreat or prevent osteoporosis caused by bacterial infections of bone,molecules comprising epitopes of known bacteria are used, as discussedabove. Where it is desired to treat or prevent osteoporosis caused byprotozoal infections of bone, molecules comprising epitopes of knownprotozoa are used. For example, such antigenic epitopes may be preparedfrom protozoa including, but not limited to, leishmania, kokzidioa, andtrypanosoma. Where it is desired to treat or prevent osteoporosis causedby parasitic infections of bone, molecules comprising epitopes of knownparasites are used. For example, such antigenic epitopes may be fromparasites including, but not limited to, chlamydia and rickettsia.

C. Method of Treating Osteoporosis

Osteoporosis caused by infectious diseases of bone that can be diagnosedusing a chaperon molecule marker can be caused by bone infectious agentsincluding, but not limited to, viruses, bacteria, fungi, protozoa andparasites, as discussed above. The method of treating osteoporosisdisclosed herein generally includes administering to a mammalian subjecta drug composition effective to treat the infectious agent causing theosteoporosis. The drug composition generally contains a drug andoptionally a drug delivery carrier and/or one or more biocompatibleexcipients. Exemplary drug delivery carriers are liposomes, micro ornanoparticles formed of natural or biodegradable synthetic polymers suchas polylactic acid, polyglycolic acid, polyhydroxyalkanoates, natural orchemically modified starches, chitosan, and proteins such as gelatin. Inthe case wherein the marker for osteoporosis is a chaperon molecule suchas a HSP, the drug composition can include one or more complexes formedof the HSP with another molecule.

The therapeutic reagents can be essentially the same as the diagnosticreagents, purified and prepared according to GMP standards. Modes ofadministration include but are not limited to subcutaneously,intramuscularly, intravenously, intraperitoneally, intradermally ormucosally.

The therapeutic regimens and pharmaceutical compositions disclosedherein may be used with additional immune response enhancers orbiological response modifiers including, but not limited to, thecytokines IFN-.alpha., IFN-.gamma., IL-2, IL-4, IL-6, TNF, or othercytokine affecting immune cells. The complexes of the HSP and antigenicmolecule are administered in combination therapy with one or more ofthese cytokines.

Drug solubility and the site of absorption are factors which should beconsidered when choosing the route of administration of a therapeuticagent. In an embodiment, HSP-antigenic molecule complexes may beadministered using any desired route of administration. Advantages ofintradermal or mucosal administration include use of lower doses andrapid absorption, respectively. Advantages of subcutaneous orintramuscular administration include suitability for some insolublesuspensions and oily suspensions, respectively. Mucosal routes ofadministration include, but are not limited to, oral, rectal and nasaladministration. Preparations for mucosal administrations are suitable invarious formulations as described below.

If the complex is water-soluble, then it may be formulated in anappropriate buffer, for example, phosphate buffered saline or otherphysiologically compatible solutions, preferably sterile. Alternatively,if the resulting complex has poor solubility in aqueous solvents, thenit may be formulated with a non-ionic surfactant such as Tween, orpolyethylene glycol. Thus, the compounds and their physiologicallyacceptable solvates may be formulated for administration by inhalationor insufflation (either through the mouth or the nose) or oral, buccal,parenteral, or rectal administration.

For oral administration, the pharmaceutical preparation may be in liquidform, for example, solutions, syrups or suspensions, or may be presentedas a drug product for reconstitution with water or other suitablevehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The pharmaceuticalcompositions may take the form of, for example, tablets or capsulesprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinized maize starch,polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets may be coated by methodswell-known in the art.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example, as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt. Liposomes and emulsions are well known examplesof delivery vehicles or carriers for hydrophilic drugs.

For administration by inhalation, the compounds for use can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

D. Resistance To Osteoporosis Induced by Bacteria or HSPs

Osteoporosis caused by bacterial infection or bone diseases can betreated by immunizing by administering to a mammalian subject animmunogenic composition. In one embodiment, the immunogenic compositionmay comprise an attenuated or modified infectious agent. In anotherembodiment, the immunogenic composition may include a HSP. Immunizationto bone diseases by HSPs are well documented. For example, in ratadjuvant arthritis (AA), resistance could be induced by immunizationwith mycobacterial HSP 60 in incomplete Freund's adjuvant (IFA). Similarfindings were obtained with mycobacterial and Escherichia coli HSP 70and HSP10, and DNA vaccination with mycobacterial HSP 60 was alsoprotective. Bardwell, J. C. A., et al., Proc. Natl. Acad. Sci. USA,81:848-852 (1984); Greenberg, S. G., J. Neuroscience, 5:1239-45.Immunization with a recombinant vaccinia virus expressing eithermycobacterial or human HSP 60 was found to suppress AA, even whenadministered after disease induction. For mycobacterial HSP 60, oraladministration was also shown to inhibit rat AA; in this case, there wasevidence for the induction of transforming growth factor(TGF-beta)-producing cells that suppressed proliferative responses tothe disease eliciting mycobacterial antigen from Mycobacteriumtuberculosis (Mt), thus pointing to a potential mechanism of diseaseresistance. Irby, R. B. et al.

TABLE 3 HSPs and protection in autoimmune disease models* ModelAnimal^(a) HSPs Route Effect Adjuvant R MtHSP 60, SubcutaneousPrevention arthritis HSP 70, HSP10 Streptococcal cell R MtHSP 60Subcutaneous Prevention wall arthritis Avridine arthritis R MtHSP 60,Subcutaneous Prevention HSP 70 Adjuvant R MtHSP 60 Vaccinia Treatmentarthritis Adjuvant R HumHSP 60 Vaccinia Treatment arthritis Adjuvant RMtHSP 60 Oral Prevention arthritis Adjuvant R MlHSP 60 Naked DNAPrevention arthritis NOD diabetes M MtHSP 60 Subcutaneous Prevention NODdiabetes M HumHSP 60 Subcutaneous Prevention, peptide treatment Pristanearthritis M MtHSP 60 Subcutaneous Prevention Collagen arthritis M MtHSP60 Subcutaneous Prevention Collagen arthritis R MtHSP 70 SubcutaneousPartial prevention Yersinia arthritis R Mtsp 60 Subcutaneous PreventionEAE R MtHSP 60 Subcutaneous Prevention peptide

Preimmunization using HSP 60 has been found to induce resistance toautoimmunity in other arthritis models, such as streptococcalcell-wall-induced arthritis-8 and, importantly, nonmicrobially inducedmodels such as pyridine-9 (a synthetic nonimmunogenic lipoidal amine) orpristane-induced arthritis, as well as collagen-induced arthritis.Similarly, development of experimental autoimmune encephalomyelitis(EAE) in rats and diabetes in nonobese diabetic (NOD) mice has beenfound to be inhibited by prior immunization with HSP 60. Therefore,immunologic exposure to HSPs could lead to resistance against variousforms of induced or spontaneous autoimmunity.

The reports on the basis of the various models collectively indicatethat resistance to autoimmunity induced by HSP 60 operates irrespectiveof the actual autoimmune disease trigger and that the suppressive effectis due to the induction of anti-inflammatory T cells responding tostress-(inflammation) upregulated self-HSP. The upregulated expressionof self-HSP 60 in the inflamed joints has been shown both in theexperimental models and in humans. HSP 60-mediated protection isdocumented in NOD diabetes, and upregulated expression of self-HSPoccurs in inflamed islets, thus it seems that self-HSP-specificanti-inflammatory T cells can also generate similar disease-suppressiveactivity in this model. Apparently, such suppressive activity iseffective in conditions as diverse as CD4⁺ T helper 1 (Th1)-mediatedchronic joint inflammation and the primarily CD8⁺ T-cell-mediateddestruction of insulin-producing T-cells. As such, recognition ofself-HSP molecules can be an important immunological strategy thatcontributes to establishment or maintenance of self-tolerance. The samecan be true in the case of inflammation caused by infection, allowingone to logically conclude that the T-cell response to HSPs operates inthe control and downmodulation of inflammatory responses, irrespectiveof their origin.

Immune Responses to HSPs in Arthritis Patients

Immune reactivity toward HSPs has been extensively investigated. Forexample, when monitoring T-cell proliferative responses to HSP 60 inchildren with juvenile rheumatoid arthritis (JRA), responses were foundto be present exclusively in patients with spontaneously remittingoligoarticular forms (OA-JRA) of the disease but not in patients withprogressive (polyarticular or systemic) forms of the disease. Suchresponses showed a pattern of fluctuation that suggested they coincidedwith development of remission, i.e. with disease suppression. Analysisof the T cells in these patients revealed the production of interleukin4 (IL-4) and TGF-beta and overexpression of CD30 upon activation withHSP 60, indicating a Th2-type response. Furthermore, upregulated mRNAlevels for IL-4 and IL-10 in the synovium of such HSP 60-responsivepatients were observed by reverse transcriptase polymerase chainreaction (RT-PCR).

The majority of such HSP 60-reactive T cells has been found to respondnot only to the mycobacterial HSP 60 molecule but especially to thehuman HSP 60. The presence of such Th2-type cells in the self-remittingforms of the disease suggests the protective nature of these T cells inhumans. Various studies have reported on immune reactivity towards HSPsin adult rheumatoid arthritis (RA) patients. Although both humoral andT-cell-mediated responses have been observed, it does not seem thatimmune responses to HSPs are a general and dominating feature in RA;certainly not in advanced or progressive forms of the disease. However,in an earlier report, it was clearly documented that an RA patient whodeveloped a self-HSP 60 crossreactive T-cell response had a rapidlyremitting course of disease. A recent study of T-cell proliferativeresponses to HSPs in adult RA patients revealed that responses to humanHSP 60 were raised upon adding IL-4 in vitro. As this was not observedfor responses to mycobacterial HSP 60, it seems that, in agreement withthe findings in OA-JRA, recognition of the human (self) moleculespreferentially triggers T cells with a regulatory phenotype. Co-cultureexperiments, in which human HSP 60-specific T-cell lines from RApatients were added to autologous peripheral blood mononuclear cells,have confirmed the regulatory nature of these T cells, as they werefound to inhibit tumour necrosis factor alpha production in mononuclearcells derived from RA patients.

HSPs are targets for regulatory T-cell responses. HSPs have uniquecharacteristics which seem to give HSPs a critical immunological status,especially their stress-dependent differential expression. Althoughstrong constitutive expression of HSP 60 has been shown in the thymicmedullary epithelium, peripheral T-cell responses to self-HSP areabundant. Thus, self-HSP-specific T cells are probably positivelyselected and subsequently escape from negative selection, suggestingtheir receptor has a low affinity for the self-HSP molecule. In theperiphery, the level of constitutive expression of HSPs, such as HSP 60,is low and peripheral tolerance for these self-antigens is likely to beless tight than for other, more abundantly available, self-proteins.Under conditions of inflammatory stress, HSP synthesis is grosslyupregulated, providing the immune system with a target through which tomonitor and control dangerous or potentially deleterious inflammatoryconditions.

Several of the characteristic features of HSPs are important.

First, their unique degree of evolutionary conservation provides themolecular basis for the demonstrated crossrecognition of microbial andself-HSP by immune cells. Second, microbial HSPs are highly immunogenicand healthy individuals have self-HSP-reactive T cells.

Third, HSPs in any cell type, everywhere in the body, respond to astress by immediate upregulation. Although some of these features mayindividually be true for other proteins in nature, the combination ofthe three features are unique for HSPs.

Mechanisms Leading to Regulation by HSP-Reactive T Cells.

A number of possible mechanisms may contribute to the regulatoryphenotype of self-HSP-reactive T cells at sites of inflammation. Suchmechanisms are proposed to be related to the peripheral tolerancemechanisms that are responsible for the persistence and safe containmentof self-HSP-reactive T cells in the immune system. First, it is possiblethat owing to low levels of self-HSP expression in peripheral tissues,self-HSP-specific T cells will simply ignore self-HSP molecules. Onlyafter exposure to microbial HSP, in infection or at the gut mucosa, willthese T cells with cross-specificity for conserved microbial HSPepitopes be stimulated and expanded. In the case of autoimmuneinflammation, self-HSP expression is upregulated and locally respondingT cells will be engaged in low-affinity interactions with self-HSPepitopes. This will lead to a downregulatory IL-4, IL-10, TGF-beta (Th2)phenotype in these cells, producing bystander regulation.

Second, it is possible that the low-level expression of self-HSPepitopes by nonprofessional antigen-presenting cells (APCs) ornonactivated APCs in the periphery, or the conserved microbial HSPepitopes in the ‘tolerizing’ gut environment, is continuously noted by Tcells under normal conditions. This recognition could skew such T cellstowards a regulatory phenotype or anergy. Subsequent involvement ofthese cells at the site of inflammation may promote their regulatoryactivity, following recognition of overexpressed HSPs on professionalAPCs. Recently, it has been demonstrated that anergic T cells, generatedwith antigen in the absence of professional APCs, exerted bystandersuppression on the proliferative responses of other T cells in thepresence of APC, provided the antigen recognized by the anergic cell waspresent in the culture. In the case of HSPs, this could mean thatquiescent HSP-specific T cells focus their regulatory activity to sitesof inflammation where HSPs become temporarily overexpressed. Duringinfection, the activity of such anergic regulators would be outweighedby a dominant frequency of T cells responding (vigorously) tononconserved microbial HSP epitopes, as well as other microbial epitopesrecognized by T cells that are not ‘silenced’ elsewhere in theperiphery.

A third possibility is that self-HSP epitopes are perceived by T cellsas APLs or closely related ‘partial agonistic’ variants of ‘fullagonist’ microbial HSP epitopes. APLs do not fully activate T cells butdo have the capacity to trigger certain effector functions such as theproduction of regulatory cytokines. This could be a profitable strategyin the case of HSPs, since exposure to full agonist microbial HSPepitopes in the gut or during infection would expand the self-HSP orAPL-oriented (regulatory) repertoire. During autoimmune inflammation,upregulated self-HSP would serve as the APL inducing a regulatoryphenotype in HSP-reactive cells. Although APLs in general may haveunpredictable and diverse effects, the fact that in this model theAPL-like self-HSP is supposed to be involved in thymic positiveselection, generating T cells that have only low affinity interactionwith the APL, may give direction (regulatory) to this specific type ofAPL.

Stimulation of Bone Resorption by Molecular Chaperones

One class of bacterial molecular chaperone, the chaperoning, wererecently discovered to be potent inducers of bone resorption (Nair, etal., Calcif Tissue Int 64(3):214-8 (1999). To address the question ofwhether the osteolytic activity of the chaperonins is unique to thisprotein class, or is a common attribute of molecular chaperonesgenerally, a number of bacterial and mammalian molecular chaperones havebeen examined for activity in the murine calvarial bone resorptionassay. All the E. coli molecular chaperones (groEL, groES, and dnaK)have been found to be active. The osteolytic activity of groEL wasinhibited by indomethacin and the natural antagonist of interleukin-1receptor antagonist (IL-1ra) but was unaffected by neutralization oftumor necrosis factor (TNF) or inhibition of 5-lipoxygenase. Mammalianmolecular chaperones of molecular mass 27, 47, 70, and 90 kDa were alsotested and, with the exception of the 47 kDa protein, all showedactivity in the murine calvarial assay. Molecular chaperones appear,therefore, to have the capacity to modulate the cellular processes inbone explant cultures, resulting in resorption of the calcified matrix.

The methods of treating bone diseases using HSP complexes disclosedherein also encompass adoptive immunotherapy. The HSP complexes can beused to sensitize antigen presenting cells (“APC”) and/or macrophagecells. The methods of using Hps complexes to sensitize macrophage and/orAPC have been described by U.S. Pat. No. 5,985,270 to Srivastava. Themethod of adoptive immunotherapy as disclosed therein is thus fullyincorporated herein by reference. The APC can be selected from amongthose antigen presenting cells known in the art, including but notlimited to macrophages, dendritic cells, B lymphocytes, and acombination thereof, and are preferably macrophages. The HSPcomplex-sensitized APC may be administered concurrently or before orafter administration of the HSP-antigenic molecule complexes. Adoptiveimmunotherapy disclosed herein allows activation of antigen presentingcells by incubation in vitro with HSP-antigenic molecule complexes.Preferably, prior to use of the cells in vivo, measurement of reactivityagainst the bone infectious agent in vitro is done. This in vitro boostfollowed by clonal selection and/or expansion, and patientadministration constitutes a useful therapeutic/prophylactic strategy.

The methods of treating and/or preventing osteoporosis include elicitingan immune response in an individual in whom the treatment or preventionof osteoporosis is desired by administering to the mammalian subject acomposition which includes an effective amount of an anti-bacteriacomposition or a HSP complex. The HSP complex is essentially an HSPnoncovalently bound to an antigenic molecule using any convenient modeof administration in combination with the adoptive immunotherapy methodsdisclosed herein. Modes of administration include but are not limited tosubcutaneously, intramuscularly, intravenously, intraperitoneally,intradermally or mucosally.

Therapeutic Dosages

Drug doses are provided in milligrams per square meter of body surfacearea because this method rather than body weight achieves a goodcorrelation to certain metabolic and excretionary functions (Shirkey, H.C., 1965, JAMA 193:443). Moreover, body surface area can be used as acommon denominator for drug dosage in adults and children as well as indifferent animal species as indicated below in Table 1 (Freireich, E.J., et al., 1966, Cancer Chemotherap. Rep. 50:219-244).

TABLE 4 Representative Surface Area to Weight Ratios (km) for VariousSpecies¹ Body Weight Surface Area Species (kg) (Sq m) km Factor Mouse0.02 0.0066 3.0 Rat 0.15 0.025 5.9 Monkey 3.0 0.24 12 Dog 8.0 0.40 20Human, Child 20 0.80 25 Adult 60 1.6 37Example: To express a mg/kg dose in any given species as the equivalentmg/sq m dose, multiply the dose by the appropriate km factor. In anadult human, 100 mg/kg is equivalent to 100 mg/kg×37 kg/sq m=3700 mg/sqm¹ Freireich, et al., 1966, Cancer Chemotherap. Rep. 50: 219-244.

Dosages of the purified complexes of HSPs and antigenic molecules usedfor administration are preferably much smaller than the dosagesestimated by the prior art methods described above. For example,according to a preferred embodiment of the methods disclosed herein, anamount of HSP 70- and/or gp 96-antigenic molecule complexes isadministered subcutaneously that is in the range of about 10 microgramsto about 600 micrograms for a human patient, the more preferred humandosage being the same as used in a 25 g mouse, i.e., in the range of10-100 micrograms. The preferred dosage for HSP-90 peptide complexes ina human patient provided by the methods disclosed herein is in the rangeof about 50 to 5,000 micrograms, the more preferred dosage being 100micrograms. Alternatively, in a specific embodiment, an amount of HSP70- and/or gp 96-antigenic molecule complexes is administeredintradermally or mucosally that is in the range of about 0.1 microgramsto about 60 micrograms for a human patient. In another specificembodiment, the therapeutically effective amount of HSP 70- and/or gp96-antigenic molecule complexes is less than 10 micrograms, e.g., in therange of 0.1 to 9 micrograms; the preferred human dosage beingsubstantially equivalent to or smaller than the dosage used in a 25 gmouse, e.g., in the range of 0.5 to 2.0 micrograms. The preferred dosagefor HSP 70-antigenic molecule complexes for intradermal or mucosaladministration to a human patient is in the range of about 5 to 500micrograms. In a specific embodiment, the therapeutically effectiveamount of HSP 70-antigenic molecule complexes is less than 50micrograms, e.g., in the range of 5 to 49 micrograms; the preferreddosage being in the range of 5 to 40 micrograms.

In one embodiment, the dosages are administered every other day for atotal of five injections. In a preferred embodiment, the doses red aboveare given once weekly for a period of about 4 to 6 weeks, and the modeof administration is preferably varied with each administration. In apreferred example, each site of administration is varied sequentially.Thus, by way of example and not limitation, the first injection may begiven intradermally on the left arm, the second on the right arm, thethird on the left belly, the fourth on the right belly, the fifth on theleft thigh, the sixth on the right thigh, etc. The same site may berepeated after a gap of one or more injections. Also, split injectionsmay be given. Thus, for example, half the dose may be given in one siteand the other half in another site on the same day.

After 4-6 weeks, further injections are preferably given at two-weekintervals over a period of time of one month. Later injections may begiven monthly. The pace of later injections may be modified, dependingupon the patient's clinical progress and responsiveness to theimmunotherapy. Alternatively, the mode of administration is sequentiallyvaried, e.g., weekly administrations are given in sequence intradermallyor mucosally.

The above regimens for administration of HSP-antigenic moleculecomplexes may occur before, during or after administration of theHSP-antigenic molecule complex-sensitized APC. For example, the mode oftherapy can be sequentially varied, e.g., HSP-antigenic moleculecomplexes may be administered at one time and HSP-antigenicmolecule-sensitized APC another time. Alternatively, HSP-antigenicmolecule complexes may be administered concurrently with HSP-antigenicmolecule-sensitized APC. Preferably, the APC and complexes areadministered to the patient within 1 week of each other.

Kits of the compositions disclosed herein include in a first container apharmaceutical composition comprising a complex of a HSP noncovalentlybound to an antigenic molecule and a pharmaceutically acceptablecarrier; and in a second container antigen presenting cells.HSP-antigenic molecule complexes may be formulated into pharmaceuticalpreparations for administration as described above. Compositions mayinclude a compound formulated in a compatible pharmaceutical carrierprepared, packaged, and labeled for treatment of the indicated boneinfectious disease. Alternatively, pharmaceutical compositions may beformulated for treatment of appropriate bone infectious diseases.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

Kits for carrying out the therapeutic regimens disclosed herein are alsoprovided. Such kits comprise in a first one or more containerstherapeutically or prophylactically effective amounts of theHSP-antigenic molecule complexes, preferably purified, inpharmaceutically acceptable form; and in a second container thesensitized APC, preferably purified. The HSP-antigenic molecule complexin a vial of a kit may be in the form of a pharmaceutically acceptablesolution, e.g., in combination with sterile saline, dextrose solution,or buffered solution, or other pharmaceutically acceptable sterilefluid. Alternatively, the complex may be lyophilized or desiccated; inthis instance, the kit optionally further comprises in a container apharmaceutically acceptable solution (e.g., saline, dextrose solution,etc.), preferably sterile, to reconstitute the complex to form asolution for injection purposes.

In another embodiment, a kit disclosed herein further comprises a needleor syringe, preferably packaged in sterile form, for injecting thecomplex, and/or a packaged alcohol pad. Instructions are optionallyincluded for administration of HSP-antigenic molecule complexes by aclinician or by the patient.

The methods and the compositions and the use thereof an be betterunderstood by the following non-limiting examples.

Example 1 Experimental

Bone samples obtained from healthy and compromised subjects were weighedand transferred to 50 ml Falcon tubes. Phosphate buffer saline (PBS)were added to the bone samples to obtain a weight (g) to volume (ml)ratio of 0.4±0.2. Samples were agitated and partially ground using the“Tissue Tearer” at rotor setting 1 (4500-8000 rpm) for 1 minute androtor setting 3 (12,000-17,000 rpm) for another minute. To increaseextraction efficiency, the bone samples were stored in Falcon tubes for9 days at 4° C. Lipids and large debris were avoided as 1 ml of eachsample are transferred to an Eppendorf tube. Any debris were cleared outby centrifuging the samples at 15,000 rpm for 10 minutes.

The samples were analyzed using first SDS-PAGE, then the Protein 200Assay on the Agilent 2 100 Bioanalyzer. Some samples that did notproduce satisfactory results (often indicated by a missing upper marker)were desalted and rerun on the Bioanalyzer. Samples were desalted byusing YM-3 Centricon tubes spun at 4200×g for approximately 1.5 hours.The Centricon filters were inverted and spun at 1000×g for 3 minutes.The samples were reconstituted to their original volume with 22 mM Tris.

Results

A SDS-PAGE was conducted on bone extracts from a control healthypatient. Slab gel electrophoresis of samples against a ladder of knownproteins suggested good separation. The Protein 200 assay was then usedwith the Bioanalyzer with a series of extracts. The results show thestronger presence of bands in the 45-65 kDa range for samples obtainedfrom compromised patents. Because the to presence of salts is known tointerfere with the assay and that the extracts used in this study wereproduced in phosphate buffer saline solution, a follow-up experiment wasdesigned to address the potential salt problem. First, a filtration stepwas added to remove salts from the samples. Furthermore, extraction wasconducted with deionized water, instead of with PBS. The changes yieldedan improved separation, and the upper marker became visible in some ofthe samples.

This study provided two methods for identifying proteins in boneextracts: the traditional SDS-PAGE gel electrophoresis and the Protein200 Chip Assay. The latter technique is more sensitive than the formerand this could be beneficial in detecting proteins produced at lowlevels. The data indicates there is an association between compromisedbone sampler and electrophoretic bands in the 45-65 kDa region.

Immunoblotting

Samples separated on one- or two-dimensional SDS-PAGE will beelectroblotted onto hnmobilon P. polyvinyldifluoride membranes(Millipore Corp., Bedford, Mass.) overnight (Laemmli, 1970). Membraneswill be washed with PBS containing 0.1% Triton X-100 (SigmanImmunochemicals) (PBS-T) and blocked with PBS-T containing 2% FCS(blocking buffer) SeraLab). Blocked membranes will then be incubatedwith the test antibody (in blocking buffer) for 1 hr. and washed withPBS-T. Bound test-antibody (anti-mouse) IgG (gamma-chain specific)(Sigma Immunochemicals) at 1:1,000 in PBS-T2% FCS. After a final washthe blots will be developed with a solution of 1 mg/ml 3,3prime-diaminobenzidine tetrahydrochloride (Sigma Immunochemicals) in 50mM Tris (Sigma Immunochemicals), pH 7.6, containing 150 mM NaCl (BDI-I)and 0.05% hydrogen peroxide (Sigma Immunochemicals). Each reaction willbe terminated by extensive rinsing with distilled water.

Protein Purification

Crude BS will be fractionated at 4° C. on a Q-Sepharose anion exchangecolumn (50 cm times 1.6 cm). The column will be equilibrated in 20 mMTris-HCl, pH 8.5 (buffer A), and the BS (generally 100400 mg) will beloaded on in the same buffer. The column will be washed with 500 ml ofbuffer A and then eluted with a 1,000-ml linear gradient of 0-1 M NaClin buffer A. Ten-ml fractions will be collected, and the absorbence at280 nm will be monitored. The location of the osteolytic chaperoneprotein will be determined by a combination of activity assay, SDS-PAGE,and the Western blot analysis. Fractions containing osteolytic activitywill be dialyzed against deionized water to remove salt and lyophilized.The fraction with the highest specific activity and the least number ofprotein bands on SDS-PAGE will then be further fractionated at roomtemperature on a second anion exchange column.

The purity of the fractions will again be assessed visually by SDS-PAGEand 100 pg of the cleanest fraction will be dialyzed against 50 mM ofTris buffer, pH 7.6 containing 10 mM MgC12 (buffer C). This sample willbe run on a 5-ml ATP-Sepharose (Sigma Immunochemicals) column. Thecolumn will be washed with 10 column volumes of butter C and boundprotein eluted in 5 column volumes of a5 mM ATP (Sigma Immunochemicals),also in buffer C. Protein will be located by SDS-PAGE and visualizedusing a silver stain kit (Sigma Immunochemicals). Gel filtration will beused to determine the molecular mass range of the osteolytic chaperoneprotein isolated by ATP-affinity chromatography. This will be done byrunning the purified protein on a Bio-Sil TSK250 (Bio Rad Laboratories)column in 0.1 M sodium phosphate buffer, pH 6.7, and measuringabsorption at 205/280 nm.

Specific Aim #3: Data Analysis and Correlation to Clinical OutcomeVariables

The experimental findings and clinical bone densitometry and Q-CTmeasurements will be documented with the use of a standardizedrelational computer database using a numerical code system. The computerprogram SPSS/PC+9.0 (SPSS Inc., 44 N. Michigan Ave., Chicago, Ill.60611) will be used for statistical analysis of the compiled data.Descriptive statistics of the raw data will be done with the use offrequency tables. Normal distribution will be determined by fitting tonormality and by obtaining normal probability plots, where the rankedobserved residuals (deviation from the mean) are plotted on the x-axisagainst the standardized values of the normal distribution on they-axis. Normal distribution is indicated if the observed residuals fallonto the straight line. Significant differences between means wereevaluated with the use of a T-test for dependent and independentsamples, and one-way ANOVA analysis of variance. In case of astatistically significant F-Test from an ANOVA using multivariatevariables, the contributing means will be differentiated by post-hoccomparison using a Tukey HSD test for unequal sample sizes. The use ofthe latter two tests will allow to determine the occurrence of thevarious Chaperone Molecules and the presence of an osteopenic bonesample according to the patients age and gender and other concomitantmedical conditions. In essence, this test will allow us to identify thepredominant chaperone molecule correlating with osteopenia.

A cross tabulation method will be used to measure the relationshipsbetween osteopenia, age, gender, other contributing factors, and thepresence of chaperone molecules by generating multiple-way frequencytables. For each possible combination of these variables, these tablesyield a cell frequency, i.e., the number of cases in the patientpopulation that had this particular combination. In addition, the crosstabulation method will allow to evaluate the reliability of the test, inother words, the statistical significance of these relationships. Hence,the presence of osteopenia, age, gender, other contributing factors suchas medical conditions are categorical variables. Multiple simultaneousrelations and interactions between the variables of the multiplewayfrequency table will be examined on the basis of log-linear equations,which allow computation of the cell frequencies that would have beenexpected if the variables involved were unrelated. This will be donewith the use of an interactive proportional fitting procedure. Thus,significant deviations of the observed from the expected frequencieswill reflect a statistically significant relationship between a specificchaperone molecule and osteopenia. Significance testing of deviations ofthe observed from the expected frequencies will be done via a PearsonChi-square test. The residual frequencies will be calculated bysubtracting the expected frequencies from the observed frequencies. Ifno relationship exists, all residual frequencies are expected to consistof positive and negative values of similar magnitude and to be evenlydistributed across the cells of the frequency table. Plotting theresiduals is therefore used as another means of assessing correlationsbetween the presence of chaperone molecules with osteopenia. In allstatistical tests employed in this study, a significance level of p<0.05will be chosen.

1. A method of detecting osteoporosis in a mammalian comprising: a)obtaining a sample of a bone related tissue or cells; and b) measuringthe concentration of at least one marker selected from the groupconsisting of infectious agents, infectious agent produced factors, andheat shock proteins (HSPs).
 2. The method of claim 1 further comprisingcomparing the concentration of a first assay with concentrations of asecond or more assays from the same individual over a period of time oragainst a standard concentration.
 3. The method of claim 1 wherein thebone related tissue or cells are obtained under conditions that do notinduce a HSP response in the mammalian subject.
 4. The method of claim 3wherein the HSP is selected from the group consisting of HSP 70, HSP 60,HSP 90, gp 96, cpn1O, cpn2O, ubiquitin, and cpn
 30. 5. The method ofclaim 2 wherein the time period between the first assay and the secondassay is at least about 12 hours.
 6. The method of claim 1 wherein thesample comprises bone cells or body fluid.
 7. (canceled)
 8. The methodof claim 3 wherein the HSP is HSP
 70. 9-11. (canceled)
 12. The method ofclaim 1 wherein the pathogen is selected from the group consisting ofbacteria, viruses, protozoa, parasites and fungi.
 13. The method ofclaim 1 wherein the pathogen is selected from the group consisting ofbacterial produced factors, viral produced factors, protozoal producedfactors, parasitic produced factors and fungal produced factors.
 14. Themethod of claim 12 wherein the bacteria is Escherichia coli. 15-19.(canceled)
 20. A method of treating or preventing osteoporosis caused byan infectious agent, an infectious agent produced factor, or a bonedisease comprising administering to a mammalian subject atherapeutically effective amount of a formulation selected from thegroup consisting of an HSP antigenic formulation and an infectious agentantigenic formulation.
 21. The method of claim 20 wherein the bonedisease is induced by bone infectious agents selected from the groupconsisting of viruses, bacteria, fungi, protozoa and parasites.
 22. Themethod of claim 20 wherein the HSP is complexed with an antigenicmaterial or formulated in combination with an adjuvant.
 23. The methodof claim 20 wherein the antigenic material is a peptide or a proteinhaving an antigenic determinant of a virus, bacteria, fungi, protozoa orparasite that induces a bone disease.
 24. The method of claim 21 whereinthe antigenic material includes an antigenic determinant of a virusselected from the group consisting of immunodeficiency virus type I(HIV-1), human immunodeficiency virus type II (HIV-II), hepatitis typeA, hepatitis type B, hepatitis type C, influenza, Varicella, adenovirus,herpes simplex type I (HSV-I), herpes simplex type II (HSV-II),rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytialvirus, papilloma virus, papova virus, cytomegalovirus, echinovirus,arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus,rubella virus and polio virus.
 25. The method of claim 20 wherein theHSP is selected from the group consisting of HSP 60, HSP 70, HSP 90, gp96, cpn 10, cpn 20, ubiquitin, cpn 30, and combinations thereof.
 26. Themethod of claim 20 wherein the osteoporosis is osteopenia.
 27. Themethod of claim 20 wherein the osteoporosis is caused by a bacteria or abacteria produced factor.
 28. A kit for use in the method of claim 1.29. A pharmaceutically acceptable composition for administration to apatient for use in the method of claim 20.